To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus Ehrlichia. Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.Ehrlichia species are known as important pathogens of medical, as well as veterinary, importance. They are intracellular microorganisms residing within the cytoplasmic vacuoles of monocytes, granulocytes, or platelets of humans and animals. Ehrlichia species elicit illnesses with fever, headache, leukopenia, and thrombocytopenia (9, 13). The genus Ehrlichia is divided into three distinct genogroups based on the similarity of the nucleotide sequence of the 16S rRNA gene: the Ehrlichia canis group (including E. canis, E. chaffeensis, E. muris, and E. ewingii), the E. phagocytophila group (E. phagocytophila, E. equi, human granulocytic ehrlichiosis [HGE] agent, and E. platys), and the E. sennetsu group (E. sennetsu, E. risticii, and the Stellantchasmus falcatus [SF] agent) (2, 15). The members of the E. sennetsu group are transmitted by aquatic vectors (16). In contrast, the members of the E. canis group and the E. phagocytophila group are both tick-borne organisms (9,15,16).